Mammalian hosts and their intestinal microbiota have co-evolved over millennia. Although customarily described as commensal, the microbiota of the gut is now known to be crucial for the normal development of host immunity and for the homeostasis of the immune system. An imbalance in microbial colonization-dysbiosis-is associated with diverse inflammatory and autoimmune syndromes, such as the inflammatory bowel diseases of the lower gastrointestinal tract. During normal immunologic development and pathophysiological reactions to challenges, small molecules produced by commensal microbes, such as lipids, can play crucial roles in shaping the immune system and its responses. Bacteroides fragilis, a well-studied resident of the human lower gastrointestinal tract, exerts interesting immune-modulating biological effects in various autoimmune diseases. In germ-free mice, wild-type B. fragilis monocolonization suppresses gut natural killer T cell (NKT cell) numbers and protects the animals from NKT cell-mediated colitis, whereas a sphingolipid- knockout strain does not provide such protection. Primary results imply that unique sphingolipid mediators originated from B. fragilis can contribute to these protective mechanisms. The current proposal describes a 5-year plan of mentored research focusing on the unique sphingolipids produced by B. fragilis and other commensal Bacteroidales and assessing their ability to modulate host immunity and protect the host from excessive inflammation. This work addresses three specific aims: (1) Acquire a comprehensive sphingolipidomic map of commensal Bacteroidales and investigate biosynthesis pathways of immunoregulatory sphingolipids, (2) Chemically synthesize commensal glycosphingolipids and assess regulation of NKT cell activity and proliferation and (3) Investigate the NKT-regulatory functions of commensal glycosphingolipids during immune maturation and upon pathological challenge. In order to attaining these goals, the principal investigator will gain knowledge and experience in microbiology and immunology based on a broad understanding of analytical chemistry and lipid mediator immunology. Didactic and technical training, in conjunction with expert mentorship by Dr. Dennis Kasper (primary mentor) and a scientific advisory committee, will aid in the successful completion of the project, which represents a key step toward independence of the applicant.